Analytical approaches to food supplement analysis
Developing new LC-MS methods Comparing characteristics of RP columns
Volume 1 / Issue 1 www.sepscience.com
January 2009
reduce nitrogen costs for LC/MS. Pure and simple Parker Balston is the leading provider of Analytical Gas Systems for the LC/MS instrument market. Generators are specifically designed to meet the stringent gas requirements for all the leading LC/MS instrument manufacturers including Agilent, Thermo Scientific, Waters, Applied Biosystems, Bruker, Varian and Shimadzu. Utilising Parker’s proprietary hollow fibre membrane technology, there are more than 10,000 systems installed worldwide. Membrane technology offers some unique performance benefits for LC/MS including phthalate free nitrogen, silent operation, no moving parts and no electrical requirements. It is technology you can trust.
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separation science
driving analytical chemistry forwards
Analytical approaches to food supplement analysis
contents
Developing new LC-MS methods Comparing characteristics of RP columns
Volume 1 / Issue 1 www.sepscience.com
January 2009
Volume 1 / Issue 1 January 2009
Rr
feature
06
research round-up 06 Comparing separation characteristics of reversed-phase columns
07 The effect of pressure on small molecule retention using RP-UPLC
22 Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements Nicola Volpi and Francessa Maccari
08 Rapid and direct determination of pesticides in water using anionexchange chromatography with coulometric detection
08 Computer-assisted solution for
multicomponent sample identification
09 SPE and GC–microEDC multiresidue analysis of royal jelly
10 Affinity partitioning of plasmid DNA with a zinc finger protein
11 FD-LC-MS/MS in breast cancer studies
Regulars
Tl Cd
11 Improved liquid chromatography
30
chrom doctor In this months column, the chrom doctor examines best practice when developing new methods for LC-MS analysis.
— Online radioactivity detection for metabolite profiling
13 Fast GC–MS pesticide multiresidue analysis of apples
14 Sequential injection methodologies for environmental analyses
15 Simultaneous HPLC and GC analysis of
Tu
lignans in Forsythia leaves
34
technology update An overview of recent technology advances in separation science and instrumentation.
16 Extraction of amphetamines from urine using a monolithic silica disc-packed spin column and HPLC–diode array detection
18 Development of immobilized enzyme reactors for phase I drug metabolism studies
19 Bioactive compound fishing with DNASeparation Science is published by Eclipse Business Media Ltd, TMC House Two, Alvaston Business Park, Middlewich Road, Nantwich, CW5 6PF, UK. Copyright 2009 Eclipse Business Media Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopying, recording or information storage and retrieval without permission from the publisher, Eclipse Business Media Ltd. Applications for the copyright owner’s permission to reproduce any part of this publication should be forwarded in writing to Permissions Dept, Separation Science, Eclipse Business Media Ltd, TMC House Two, Alvaston Business Park, Middlewich Road, Nantwich, CW5 6PF, UK. Separation Science does not verify any claims or other information appearing in any of the advertisements contained in the publication, and cannot take any responsibility for any losses or other damages incurred by readers in reliance on such content.
based bioseparations and chemical analysis
20 MSPD extraction of carbadox and
olaquindox in feed followed by hydrophilic interaction ultra-highpressure liquid chromatographic analysis
for research news, technical articles, product updates, jobs and applications visit. . . separation science — volume 1 issue 1
contents
3
David Hills – Scientific Director
[email protected] Peter Myers – Chief Scientific Officer
[email protected] New Year, new ideas…
S
o it’s now 2009 and Separation Science is finally off and running on a regular basis. Before going any further I’d just like to thank all of you who took the time to contact me with your thoughts on the launch issue,
whether that was in person at the ISC meeting in Münster or subsequently by telephone or email. Thankfully, the vast majority of these comments were positive and I’ve done my best to take them all on board prior to our full launch with
David Barrow University of Cardiff, UK
Melissa Hanna-Brown Pfizer, UK
Zongwei Cai Hong Kong Baptist University
Tuulia Hyötyläinen University of Helsinki, Finland
Yi Chen Chinese Academy of Sciences, Beijing, China
Gongke Li Sun Yat-Sen University, Guangzhou, China
Gert Desmet Vrije Universiteit Brussel, Belgium
Yong-Chien Ling National Tsing Hua University, Taiwan
C. Bor Fuh National Chi Nan University, Taiwan Klara Valko, GSK, UK Y.S. Fung Hong Kong University Jean-Luc Veuthey University of Geneva, Switzerland Xindu Geng Northwest University, Xi’an, China Claudio Villani Universita’ degli Studi di Roma “La Luigi Mondello Sapienza”, Italy University of Messina, Italy Cheing- Tong Yan Paul Haddad Center of Environmental Safety and University of Tasmania, Australia Hygene, Taiwan
this issue. Hopefully, you’ll notice some of these changes as you peruse the page of this digital publication. If you’d like to continue receiving Separation Science simply visit www.sepscience.com, click on the subscribe button and fill out a short form and you’ll receive the publication each month – and don’t forget, it’s absolutely free. You’ll also notice that we now offer two different formats for the magazine: the standard digital turning-page version for those of you with broadband; and a static PDF version for those of you without. The choice is up to you. By the way, when you visit www.sepscience.com, you’ll also notice a couple of changes: r5IFA"QQMJDBUJPO/PUF%BUBCBTFIBTJNQSPWFETFBSDI
scientific advisory council
functionality – as well as including over 4,000 application notes in PDF format, it is now searchable by multiple keywords
Hian Kee Lee National University of Singapore, Singapore
Edward Browne GSK, Singapore
in both AND and OR logic. r"TFBSDIBCMFKPCTEBUBCBTFmJGZPVSFMPPLJOHGPSBOFXTUBSUJO the New Year why don’t you try it out. Finally, I’d just like to pique your interest by mentioning our upcoming scientific conference – Separation Science Singapore. It will take place between 26-28 August this year at the stateof-the art Biopolis Park in Singapore, and features an array of high-quality speakers from around the world, as well as from the immediate region, covering analytical challenges in food, pharma, enviro, bioscience and energy markets. I’ll bring you more information on this exciting event in later issues but for more of a teaser go to page 29. Enjoy the issue, and as always if you have any questions or comments just send me an email. David Hills
[email protected] 4
from the editor
contacts Dean Graimes Publishing Director +44 1270 610037
David Hills Scientific Director +44 1270 610012
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separation science — volume 1 issue 1
5
Rr Research round-up
Key
Comparing separation characteristics of reversed-phase columns
Email the author
USA Silica-based chemically bonded stationary
are used for alkyl and aromatic ligands,” he
phases continue to dominate the practice of
said.
Article link
Product information
Comment
reversed-phase liquid chromatography, in spite
The key findings of the study are that
of efforts to replace them with polymeric or
for the XBridge C8, C18, C18 Shield, and
other inorganic oxide-based materials.
phenyl chemically bonded stationary
Dr Colin Poole from the Department of
phases, changes in selectivity in going
Chemistry at Wayne State University, USA,
from one phase to the other are small
looked at the differences in the system
and largely independent of the mobile
constants of the solvation parameter model
phase composition used. “Larger changes
and retention factor correlation plots for varied
are associated with weak electrostatic
solutes to study the retention mechanism
interactions and steric repulsion for a few
on XBridge C8, XBridge Phenyl and XTerra
specific compounds,” he added.
Phenyl stationary phases (all Waters Corp.,
“There is a road block in method
Milford, Massachusetts, USA) with acetonitrile–
development for those mixtures that are
water and methanol–water mobile phases
difficult to separate, in that changing the
containing from 10 to 70% (v/v) organic
column type or supplier, will rarely result
solvent. Published in Chromatographia [68
in significant progress as columns become
(7-8) 491-500 (2008)], these stationary phases
more alike. This may largely be a waste
are compared with XBridge C18 and XBridge
of effort and should be no more than a
Shield RP18 characterized in an earlier report
last resort. Older column types are more
using the same protocol.
variable and may offer a greater chance of
Dr Poole explained that improvements
success,” he said. The use of models such as
in chemically bonded stationary phases
the solvation parameter provides a means
have resulted in new products being much
to select columns with different separation
more alike than in the past. “Large changes
characteristics and to know in advance what
in retention are often due to differences in
chemical basis for a separation is being
the phase ratio, while changes in selectivity
exploited.
(relative retention) are quite small. Even changes in the structure of the bonded ligands seems to have little effect on selectivity when pure silica particles and high bonding densities 6
research round-up
www.sepscience.com
separation science
driving analytical chemistry forwards
The effect of pressure on small molecule retention using RP-UPLC UK The effect of inlet pressure on the retention of a series of low molecular
THE NEW SEPARATION SCIENCE REPORTS ARE NOW AVAILABLE...
weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a
Enviro Report Organochloride analysis using graphitized carbon black Automated Disposable Pipette Extraction of Pesticides from Fruits and Vegetables
commercial ultra-high-pressure system (Waters UPLC instrument) in the
WWEM 2008 Preview Featured Books
Journal of Chromatography A [1209 (1-2), 195-205 (2008)].
“Changes in retention with pressure, can be considerable for large
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molecules such as proteins, but are they significant for lower molecular
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weight compounds? The study is of importance as some commercial
E E E E E
0.99846 for each analyte. The accuracy and precision are good enough to make the procedure applicable to routine
separation science — volume 1 issue 1
research round-up
9
use in the analysis of residual levels of the tested pesticides and especially the residues of the synthetic acaricides used for apicultural purposes,” she said. In the future, she believes this method can be used by analytical laboratories in order to determine the presence of acaricide and other pesticide residues in royal jelly samples. “The method has already been used for assessment of residues detected in royal jelly after the application of four registered in apiculture synthetic acaricides,” she said. The next step is to assess the presence of pesticide residues in royal jelly in vivo. “Royal jelly is a rather polar matrix with a high content of water, proteins and lipids so it is possible for pesticide residues to be present,” she concluded.
Affinity partitioning of plasmid DNA with a zinc finger protein Portugal A study, published in the Journal of
in the PEG phase of a PEG 600–DEX 40 ATPS.
Chromatography A [1206 (2), 105-112 (2008)],
This represents a 1322-fold increase in the
investigated the possibility of using a DNA
protein partition coefficient in comparison
binding protein [in this case a zinc finger
with the non-PEGylated protein (Kc = 0.013).
protein (ZnFP)], as an affinity ligand in aqueous
In the presence of pDNA containing a specific
two-phase extraction of plasmid DNA (pDNA).
oligonucleotide recognition sequence, the
“Aqueous two-phase extraction has already
zinc finger moiety of the PEGylated fusion
proved to be very promising for future
protein bound to the plasmid and steered the
utilization in the large-scale preparation of
complex to the PEG-rich phase.
pDNA for DNA vaccines and gene therapy.
“We found that, in systems where isolated
However, it lacks selectivity that could be
ZnFP and pDNA accumulate in different
improved by addition of affinity ligands,” said
phases, after mixing them together the
lead researcher, Dr João Marcos from the
partition of pDNA shifts to the phase where
Department of Chemistry at the University of
the ZnFP accumulates. This was true both for
Minho in Braga, Portugal.
the case where a native and a PEGylated ZnFP
Previously, small molecules have been used
were used. The latter case is more promising as
as affinity ligands in aqueous two-phase
it selectively accumulates pDNA in the phase
extraction of proteins with excellent results.
where less protein accumulates,” Dr Marcos
However it was not known if a similar principle
explained.
could be used for pDNA given its structural
According to him, the results presented in
and size differences. Therefore, in this study
this paper show it is possible to increase the
the affinity isolation of prepurified pDNA
selectivity of aqueous two-phase systems for
from model buffer solutions using native and
pDNA extraction using a DNA binding protein.
poly(ethylene glycol) (PEG) derivatized zinc
“After this proof-of-concept paper we are
finger–GST (glutathione-S-transferase) fusion
now applying this knowledge to design and
protein was examined in PEG–dextran (DEX)
develop a purification process of pDNA based
aqueous two-phase systems (ATPSs).
on aqueous two-phase systems,” he concluded.
In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected 10
research round-up
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FD-LC-MS/MS in breast cancer studies
is our opinion that such an FD-LC-MS/MS method could clarify the
Japan
dynamic cellular processes of proteins in
Kazuhiro Imai and colleagues from Musashino
tissues and demonstrate other candidates for
University and the Japanese Foundation
molecular markers in breast cancer diagnosis.”
for Cancer Research (both Tokyo, Japan),
“Previous proteomic studies have reported
have published the results of a proteomics
individual changes in expressed proteins.
study on human breast cancer cell lines
In contrast, our work has demonstrated
using fluorogenic derivatization-liquid
the dynamic flow of signal proteins in both
chromatography/tandem mass spectrometry
normal and cancer cells, and thus highlighted
[Biomedical Chromatography, 22 (11), 1304-
presumptive mechanisms leading to invasion,
1314 (2008)].
metastasis, proliferation and inhibition of
“Previously, we developed fluorogenic
cancer cells, as well as the new biomarker
reagents possessing a benzofurazan skeleton,
candidates. Furthermore, we have suggested
such as NBD-F, which are now available
that tropomyosin-1 could play a key role in
as reagents for amines and amino acids1,”
counteracting tumour progression,” explained
began Imai, before expanding, “The current
Imai. The research team feel that the
article relates to works combining the use of these reagents with the HPLC separation of
FD-LC-MS/MS method is a highly reliable,
fluorescent derivatives.”
reproducible, sensitive and easy-to-handle
He continued, “Although several molecular
method for proteomics analysis and, in fact,
markers for diagnosis and therapy of breast
is superior in terms of understanding the
cancer exist, their versatility is limited because
performance of the dynamic events in tissues.
of the lack of direct information on dynamic
The method should be further applied to
cellular processes of proteins in tissues. To
understanding the complex cellular events in
distinguish and identify minute changes
disease states.
in the expressed proteins between cancer
1
Biomedical Chromatography, 9, 106-109 (1995).
and normal tissue, highly sensitive and reproducible proteomic analysis are required. It
Improved liquid chromatography — Online radioactivity detection for metabolite profiling Belgium “Up until this point, radiotracer technology (14C or 3H) has been the method of choice to study the in vivo disposition of a new drug as it enables the quantitative detection of the parent drug and all of its metabolites in complex matrices. Metabolites are structurally related entities and structure differences can be minor. Therefore, a complete separation between all drug-related compounds is, although necessary for quantification, difficult to achieve,” said Dr Filip Cuyckens from Global Preclinical Development at Johnson & Johnson Pharmaceutical R&D in Beerse, Belgium. In a paper he published in the Journal of Chromatography A [1209 (1-2), 128-135 (2008)], Dr Cuyckens describes the successful combination of very-high-pressure liquid chromatography (VHPLC) with online radioactivity detection (RAD) facilitated by improvements in online radioactivity detection, as well as in column loading and peak capacity. “Many researchers in our field were hoping for an efficient coupling of ultra-performance liquid chromatography (UPLC) separation with online radioactivity detection. The major issue is that the narrow peak widths obtained in separation science — volume 1 issue 1
research round-up
11
UPLC separations appear intrinsically incompatible with the relatively high counting times needed to reach adequate sensitivity in radiochemical detection,” he explained. Traditionally 500 to 1000 μL flow-through cells are used in radioactive detection of drug metabolism samples because the radioactivity concentrations in these (especially in vivo samples) are rather low. According to him, these large cell volumes are detrimental for the peak width and high-resolution separations obtained in UPLC. In order to keep a good resolution, the cell volume needs to be decreased, which in turn is detrimental to sensitivity. “In our laboratory, we developed an efficient combination of
Fast GC–MS pesticide multiresidue analysis of apples Slovakia A fast gas chromatographic–mass
UPLC with a conventional radiodetector. This combination was
spectrometric (GC–MS) method is proposed
made possible by improving the on-line radioactivity detection, as
for pesticide multiresidue analysis of apples
well as increasing the column loading and peak capacity for ultra-
in Chromatographia [68 (supplement 1),
high-pressure separations,” he said.
49-55 (2008)]. Professor Eva Matisová from
In the study, the sensitivity of 14C detection was improved
the Institute of Analytical Chemistry at the
by the use of a variable scintillation flow achieved via a simple
Faculty of Chemical and Food Technology of
modification to the classic online radiochemical detection set-
the Slovak University of Technology, Slovak
up. A modification of the flow-through cell design in which
Republic, used the QuEChERS method for
internal diameter of the tubing was reduced further increased
sample preparation and GC–MS analysis was
the sensitivity and resolution by decreasing peak tailing. The
performed with a PTV, an autoinjector, and a
injection of relatively large volumes was made possible by the use
quadrupole benchtop MS detector.
of columns packed at ultra-high-pressure with 2.2 μm particles.
“Speeding up GC analysis provides
Because of the reduced back pressure using these larger particles,
unquestionable benefits towards conventional
two 3 x 150 mm columns could be coupled, allowing four fold
GC, such as higher laboratory throughput,
larger injection volumes and a 50% improved separation at a
reduced GC operating costs, and better
similar backpressure compared with a standard 2.1 x 150 mm UPLC
analytical precision by making possible
column.
more replicate analyses. Nowadays, fast
“This is the first UPLC ¬ on-line radioactivity detection set-up that
GC can be performed on commercial gas
works in practice for samples with limited radioactivity, not only
chromatographs, which are equipped with
for standards. We are now using the approach in our lab for real
high-speed injection systems, electronic gas
projects and are replacing our existing radio-HPLC systems with
pressure control, rapid oven heating/cooling
radio-UPLC systems. The use of the larger and coupled columns
and fast detection,” Professor Matisová said.
allowing higher loadability and plate numbers is also very useful for
In the study, compounds were separated
other applications; for example, chemical impurity quantification,”
under temperature-programmed conditions
he concluded.
on a narrow-bore diphenyldimethylsiloxane column. In one chromatographic run 61 pesticides of different chemical classes, and triphenyl phosphate as internal standard, were determined in 11 min. Calibration was performed with matrix-matched standard solutions and response to the pesticides was a linear function of concentration in the range 1–500 ng/mL (equivalent to 1–500 μg/kg in real samples). High values of the determination
separation science — volume 1 issue 1
research round-up
13
coefficients (R2; 0.9900–1.0000) were obtained for most of the pesticides. Limits of detection and quantification were determined. According to Matisová, the key outcome was the development and validation of the method for multiresidue analysis of pesticides in nonfatty food (apples) by fast GC-MS with PTV injection in solvent mode and using a narrow-bore capillary column. Quadrupole MS detectors have been most widely utilized in analytical practice. “From the results obtained it can be concluded that the used quadrupole detector is fast enough for proper reconstruction of the analytes peaks and that the method can be used for multiresidue analysis,” she said. The method is applicable to pesticide residues analysis at ultra-trace concentration levels in real samples in non-fatty food. “Our future work will be directed to endocrine disrupting pesticides analysis in environmental matrices,” she added.
Sequential injection methodologies for environmental analyses Brazil Published in Analytical Chimica Acta [628(2), 123-132 (2008)] is an article by Jorge Masini and colleagues (Insituto de Quimica, Universidade de São Paulo, Brazil), describing the implementation of a stepwise solvent elution in sequential injection chromatography (SIC) for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis. “Part of my research work focuses on the development of sequential injection or flow injection methodologies for environmental analyses,” explained Masini, adding “These techniques are suitable for precise liquid handling and control of reaction times, but are limited to only a few analytes per sample injection, such that multi-analyte determinations have been a target for practitioners of flow/sequential injection techniques.” “Coupling low pressure flow systems to monolithic stationary phases has allowed us to improve the capabilities of sequential injection or flow injection techniques toward multi-analyte determinations. We were originally interested in the on-line determination of amino acids that are excreted by marine microalgae in batch cultivation. This was a goal that required multi-analyte determinations and precolumn derivatization, so sequential injection chromatography was our choice to start the method development,” he continued. The concept of sequential injection chromatography was exploited to automate the fluorimetric determination of amino acids after precolumn derivatization with o-phthaldialdehyde in the presence of 2-mercaptoethanol using a reverse-phase monolithic C18 stationary phase. Additional to the on-line precolumn derivatization, the proposed method involved five steps of isocratic elutions allowing the use of a wide range of polarities during the elution. “To our best knowledge, this simple approach had not yet been described in SIC methodologies, which had previously used only a single eluting solution (isocratic), being limited to separate components of simple mixtures, and being especially applied to the determination of pharmaceutical products. In a mix of 18 amino acids, the proposed procedure allowed us to 14
research round-up
www.sepscience.com
separate 14 analytes with resolutiongreater than1.5,” Masini explained. He concluded, “We believe that the methodology described in the referred article can extend the application of SIC to the separation of complex mixtures with potential application to physiological studies of amino acid metabolism in algae, as well as in environmental studies related to carbon and nitrogen assimilation by phytoplankton. Development and application of SIC procedures to perform on-line monitoring for adsorption/desorption of herbicides and their metabolites onto/from soil particles is another on-going project in our laboratory.”
Simultaneous HPLC and GC analysis of lignans in Forsythia leaves Hungary The biological activities of lignans are of pharmaceutical importance. Among butyrolacton lignans, arctigenin and its glucoside, arctiin occur in Forsythia species in high quantities. Éva Sedlák from the Department of Plant Anatomy at Eötvös Loránd University in Budapest, Hungary conducted a study to find the most suitable extraction method for these lignans, and identify and quantify the lignan constituents present in leaf samples of different Forsythia species and cultivars by highperformance liquid chromatography (HPLC) and gas chromatography, (GC), simultaneously. Published in Chromatographia [68 (supplement 1), 35-41 (2008)] lignans, carboxylic acids and sugars were determined by gas chromatography (GC) with mass selective detection, the lignans also by high-performance liquid chromatography with UV detection in the leaf extracts of four species and four cultivars of Forsythia plant. Three methods were used to optimize the extraction of the main lignan constituents (arctiin, arctigenin), which possess significant pharmaceutical effects. “Our results suggested that the supercritical fluid extraction applying CO2+60% methyl alcohol was the best method for obtaining the highest extraction yield of lignans,” said Sedlák. Regarding the identification and quantification of aglycone lignans, both HPLC and GC-MS proved to be efficient. To quantitate lignan glycosides, HPLC was the method of choice. According to her, from a pharmaceutical point of view the most relevant, arctigenin, was found in the highest quantity in the cultivar ‘Robusta’ of Forsythia ovata. “We want to utilize our optimized extraction and analytical techniques in further basic research and technical applications. Our final goal would be to prepare medicinal product from the highest lignan containing Forsythia ovata species,” she concluded.
separation science — volume 1 issue 1
research round-up
15
Extracting amphetamines from urine using monolithic silica disc-packed spin columns Japan To overcome the limitations of solid-phase
follows: linear curves (drug concentrations of
extraction, Dr Akira Namera and colleagues
0.2–20 μg/mL); correlation coefficients >0.99;
from the Department of Forensic Medicine
detection limit, 0.1 μg/mL.
at Hiroshima University, Japan, developed
“In our previous study, we packed monolithic
a device comprising a spin column packed
silica into a capillary glass tube (i.d. = 0.2 mm)
with octadecyl silane-bonded monolithic
and the extraction device was created by
silica for extracting amphetamines and
connecting a microsyringe to the capillary
methylenedioxyamphetamines from
column. We demonstrated that amphetamines
urine. A paper published in the Journal of
in urine could be extracted using this device,”
Chromatography A [1208 (1-2), 71-75 (2008)]
he explained. However, sample filtration was
states that the proposed method is not only
required to avoid blockage within the tube,
useful for drugs from biological materials but
and thus, only one sample could be obtained
also highly reproducible for the analysis of
through a batch processing cycle. In order to
these drugs in urine.
overcome these problems, a monolithic silica
Liquid-liquid or solid-phase extraction
disc was packed into a spin column, wherein
(SPE) methods are widely used for extracting,
the structure of monolithic silica combined
purifying, and enriching drugs and medicines
the support body and the surface area for
from biological materials. However, these
each unit volume is wide by comparing
methods involve laborious, intensive and
with a particle-type silica. “The handling
expensive preparatory procedures. “GL
procedures such as sample loading, washing
Sciences informed me that monolithic silica
and elution of the target drugs were only
has been investigated as a new type of
exhibited by a centrifugation of the column.
separation material for high-performance
In addition, many samples can be processed
liquid chromatography (HPLC). Although
simultaneously,” he added.
the conventional materials used for SPE
According to him, this spin column has many
were similar to those used for HPLC, we
advantages: its operation procedure is simple,
think monolithic silica has potential for drug
requires a low eluate volume and does not
extraction and purification from biological
involve solvent evaporation. “It is expected
materials,” said Dr Namera.
to take the place of conventional solid-phase
Urine (0.5 mL), buffer (0.4 mL) and methoxyphenamine (internal standard) were
extraction cartridges in many fields,” he concluded.
directly added to the preactivated column. The column was centrifuged (3000 rpm, 5 min) for sample loading and washed. The adsorbed analytes were eluted and analysed by HPLC, without evaporation. The results were as 16
research round-up
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Development of immobilized enzyme reactors for phase I drug metabolism studies Switzerland Cytochrome P450 enzymes (CYP450) are the major drug-metabolizing enzyme systems in
inhibition studies were also reported. In comparison with in-solution assays, in
the human liver responsible for the oxidative
which CYP450 enzymes are known to be
conversion of approximately 90% of marketed
rapidly inactivated, the system was stable
drugs.
for performing ca. 15 experiments in 2 days,
In order to determine the CYP450 isozyme
according to him.
involved in a specific drug metabolism, in vitro
“This technique can be applied in drug
screening is usually performed by incubating
discovery for the evaluation of drug-drug
in-solution human liver microsomes or
interactions and assessment of metabolism
recombinantly expressed human CYP450
profiles of novel chemical entities. Thanks
isozymes with drugs (alone or with a potential
to the possibility to reuse the immobilized
inductor or inhibitor). These incubations
enzyme for several analyses, this strategy is
require relatively large amounts of expensive
cost-effective in comparison with conventional
recombinant enzymes. Prof. Jean-Luc
in vitro tests,” he said. To date, the team is trying
Veuthey and colleagues from the School of
to improve the stability of the IMERs, and is
Pharmaceutical Sciences at the University of
working also on a different approach which
Geneva, Switzerland have developed CYP450-
will be published in the near future.
based immobilized enzyme reactors (IMERs) to perform automated on-line phase I drug metabolism studies. Published in the Journal of Chromatography A [1206 (1), 2-10 (2008)], the major aim of this study was to prepare two IMERs containing CYP2D6 and CYP3A4 for performing automatically phase I drug metabolism. “CYP450 is a very complex enzymatic system constituted by three enzymes embedded in a phospholipidic membrane and requiring NADPH as cofactor. It was, therefore, not possible to use the covalent immobilization procedure already developed in our laboratory with trypsin,” Prof. Veuthey explained. Using an original strategy (NeutrAvidin covalent immobilization on monolithic disc; biotinylation of CYP450; coupling of the biotinylated CYP450 on the NeutrA-disc), it was possible for the team to obtain two discs (2 x 6 mm i.d.) containing immobilized CYP2D6 and CYP3A4 to perform automatically drug metabolism studies by LC-ESI-MS/MS. The method was tested with probe substrates and 18
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Bioactive compound fishing with DNA-based bioseparations and chemical analysis China According to a recent article in Biomedical Chromatography [22 (10), 1164-1172 (2008)], Professor Ping Li and researchers from the China Pharmaceutical University, Nanjing, China, have performed a screening and mechanism study of components targeting DNA from the Chinese herb Lonicera japonica by liquid chromatography/mass spectrometry and fluorescence spectroscopy. DNA is the potential molecular target of many antimicrobial, antiviral and antitumour active drugs. The screening and identification of DNA-targeting agents from natural products extracts are attractive, and the interaction of DNA with small molecules has been an intensive topic which has fascinated scientists for decades as it provides insights into rational design of drugs targeting to DNA. “As the biological separation system, DNA can be used to selectively fish for bioactive compounds from multicomponent samples. By ultrafiltration sampling and LC-MS analysis, DNA-bound compounds can be easily identified from their chromatographic fingerprints before and after interaction with DNA,” explained Li, adding “Four flavonoids were fished and identified from Lonicera japonica extract using this method. The binding mechanism of these flavonoids with DNA was then evaluated to be groove binding by fluorescence spectroscopy. For L. japonica, the flavonoids binding to DNA may be one of the mechanisms of its antimicrobial, antiviral or antitumour actions.” “Our future work will extend our bioseparation and chemical analysis strategy to other biological separation systems; for example, target protein, cell etc., for rapid and effective screening of potential bioactive compounds from complex multicomponent samples,” Li concluded.
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research round-up
19
technical articles on chromatography? updates on recent research studies?
MSPD extraction of carbadox and olaquindox in feed followed by hydrophilic interaction ultra-highpressure liquid chromatographic analysis Lithuania A new method involving matrix solid-phase dispersion
practical advice on routine analysis?
(MSPD) extraction and hydrophilic interaction ultraperformance liquid chromatography (HILIC-UHPLC)
applications of new technology?
with photodiode array detection was developed for the determination of carbadox and olaquindox in feed.
information on product developments? Described in the Journal of Chromatography A [1209 (1-2), market trends and opinions?
83-87 (2008)], the separation of carbadox and olaquindox was achieved within 1 min on the 1.7 μm Acquity UPLC BEH HILIC column using isocratic elution with a mobile phase consisting of
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10 mmol/L ammonium acetate in acetonitrile-water (95:5, v/v) at a flow rate of 0.5 mL/min. “In recent years our group has collaborated closely with the National Food and Veterinary Risk Assessment Institute
separation science driving analytical chemistry forwards
of Lithuania in developing new analytical methods for the determination of veterinary drug residues in food and feed,” said main author, Professor Audrius Padarauskas from the Department of Analytical and Environmental Chemistry at Vilnius University in Lithuania. The institute tasked his
at
group with the development of a simple, fast and reliable method for the routine monitoring of carbadox and
c n e i c s n ratio cl driiving anallyytitica
20
research round-up
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olaquindox in feeds. Optimization of MSPD extraction parameters, such as type of solid sorbent and elution solvent were performed. Optimal conditions selected for MSPD extraction were: 0.25 g of feed sample, 0.5 g of octadecylsilica as solid sorbent and 10 mL of acetonitrile-methanol (8:2, v/v) as eluting solvent. Both analytes provided average recoveries from spiked feed samples ranging from 89.1 to 98.4% with relative standard deviations less than 10%. “In my opinion, the main strength of the present work is the combination of matrix solid-phase dispersion extraction with hydrophilic interaction (HILIC) ultra performance liquid chromatographic analysis. Direct injection, without evaporation/reconstitution, of the extract onto the separation column is therefore possible, which saves time and prevents sample losses. Another significant advantage is that less polar interfering compounds requiring gradient elution by conventional reversed-phase HPLC are eluted early in an isocratic HILIC system, thereby simplifying and accelerating the overall analysis,” Professor Padarauskas explained. He believes this combination should be very promising for the analysis of polar compounds in complex biological matrices such as food, feed, plants etc.
separation science — volume 1 issue 1
research round-up
21
22
feature article — SAX-HPLC analysis of food supplements
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Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements Nicola Volpi and Francesca Maccari Department of Biologia Animale, Biological Chemistry Section, University of Modena and Reggio Emilia, Italy
The amount and quality of chondroitin sulfate (CS) from several Czech Republic food supplement/nutraceutical preparations was determined. To quantify CS, two different analytical approaches were validated [11] and applied: specific and sensitive agarose-gel electrophoresis and strong anion exchange (SAX)-high performance liquid chromatography (HPLC) determination of the constituent disaccharides after treatment with specific chondroitin lyases. The CS content in food supplement products were found to conform to the label specifications in only four of the ten analysed samples. Four of the food supplement preparations were found to contain approximately 0-1% CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approximately 30-45% of the declared CS, and one preparation was found to contain approximately 2% hyaluronic acid. SAXHPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and possible origin. The CS contained in eight food supplements resulted to be of bovine or porcine origin, one from cartilagineous fish and in one case it was not possible to determine the origin because of very low CS content. Based on these analytical results, quality of the sampled Czech Republic food supplement formulations was poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of high-quality products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high-quality products and applied by quality control laboratories to confirm the purity and label claim of CS in raw materials and nutraceuticals.
Introduction Chondroitin sulfate (CS) (Figure 1), a
hand (OA) [5-7]. Moreover, CS alone
and various nutraceutical preparations
very complex, polydisperse, natural
or in combination with glucosamine,
[9, 10], and their purity and quality
glycosaminoglycan (GAG) highly
is utilized as a dietary supplement [7,
were generally found to be
heterogeneous for relative molecular
8]. As a consequence, the number
inconsistent with the specifications
mass, charge density, structure, and
of pharmaceuticals containing CS
claimed on the product labels. In this
biological and pharmacological
has increased, as well as the types of
article, we determine the CS amount,
activities [1, 2], is recommended
formulations, including tablets and
quality and origin in several Czech
by EULAR [3, 4] as a SYSADOA
caplets, capsules, ophthalmic solutions
Republic nutraceutical samples using
[Symptomatic Slow Acting Drug for
and liquid preparations [9-11].
the same analytical methodology
osteoarthritis (OA)] drug in Europe for the treatment of knee, hip and separation science — volume 1 issue 1
Previous studies reported a determination of CS in raw materials
applied to a recent CS evaluation in US food supplements [11].
feature article — SAX-HPLC analysis of food supplements
23
Figure1
6S: ΔUA-[13]-GalNAc-6S, ΔDi-4S:
CH2OR 2 COO H O
H O
ΔUA-2S-[13]-GalNAc-6S, ΔDi-4,6diS:
O
ΔUA-[13]-GalNAc-4S,6S, ΔDi-2,4diS
H H
H
H
H
H H
O
OR 1 O
H OH
ΔUA-[13]-GalNAc-4S, ΔDi-2,6diS:
ΔUA-2S-[13]-GalNAc-4S) were from Seikagaku. High resolution agarose,
NHCOCH3
certified for molecular biology, was
OR 3
from Sigma. All other reagents were of
R1 = R2 = R3 = H: nonsulfated chonroitin R 1 = SO3 and R2 = R3 = H: chondroitin-4-sulfate, CSA R 2 = SO3 and R1 = R3 = H: chondroitin-6-sulfate, CSC R 2 = R3 = SO3 and R 1 = H: chondroitin-2, 6-disulfate, CSD R 1 = R2 = SO3 and R3 = H: chondroitin-4, 6-disulfate, CSE R 1 = R3 = SO3 and R2 = H: chondroitin-2, 4-disulfate, CSB R 1 = R2 = R3 = SO3 : trisulfated chondroitin
analytical grade. Sample preparation: All the used nutraceuticals contained various other ingredients in different amounts, in particular glucosamine, collagen, vitamins and/or MSM (Table 1).
Figure 1. Structures of disaccharides forming chondroitin sulfate.
Stock solutions (10 mg/mL) of all the Materials and Methods
analyses [12], and has a claimed CS
products were carefully prepared.
Materials: Various food supplement
content greater than 98% [12]. CS
No sample pretreatment was
samples in the form of tablets or
fractions having different molecular
performed apart from centrifugation
capsules containing CS in different
mass values for high-performance
at 10,000 RPM for 10 min to remove
formulations and amounts
size-exclusion chromatography
insoluble material, mainly derived
(Table 1) were obtained from the
(HPSEC) analysis, were prepared by
from excipients and salts present in
Czech Republic market. The European
gel-permeation and evaluated by
the preparations, and a proteolytic
Pharmacopeia CS (EPCS) reference
means of analytical ultracentrifugation
treatment to remove possible
standard, manufactured by Bioiberica
[13]. Chondroitinase ABC, chondroitin
interfering proteins. The EPCS
(www.bioiberica.com/) and approved
ABC lyase, from Proteus vulgaris
reference standard was dissolved in
in 2004 as the Chemical Reference
(EC 4.2.2.4), 0.5-2 units/mg, and
water to make authentic CS solution
Substance (CRS) by the European
chondroitinase ACII, chondroitin
(10 mg/mL).
Pharmacopeia Commission was
AC lyase, from Arthrobacter aurescens
Analytical procedures: The
utilized as a standard in these analyses.
#(EC 4.2.2.5), were from Sigma.
analytical techniques used for
Unsaturated CS/DS disaccharides
This CS standard is produced from bovine cartilage, as evaluated by HPLC
(ΔDi-0S: ΔUA-[13]-GalNAc, ΔDi-
the quantitative and qualitative evaluation of CS in food supplements
Table1
24
Sample
Declared CS Content
Formulation
A
250 mg/capsule
Glucosamine, Collagen, Saccharides
B
250 mg/tablet
Glucosamine, MSM
C
25 mg/tablet
Glucosamine, Collagen, MSM
D
200 mg/tablet
Glucosamine, MSM
E
200 mg/tablet
Glucosamine
F
67 mg/capsule
Glucosamine, Collagen, MSM, Vitamin C, Vitamin E
G
200 mg/tablet
Glucosamine, MSM
H
400 mg/tablet
Glucosamine
I
25 mg/tablet
Glucosamine, Collagen, MSM
L
75 mg/tablet
Glucosamine, Collagen
feature article — SAX-HPLC analysis of food supplements
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have been previously published in
Drug Evaluation and Research (CDER),
Results
detail [11]. In particular, agarose-
Center for Veterinary Medicine (CVM)
First, an accurate determination
gel electrophoresis and HPLC
published in May 2001 [18], including
of the theoretical CS content was
separations of the unsaturated CS
specificity, linearity, detection (LOD)
performed by evaluating real weight
disaccharides produced by the action
and quantitation (LOQ) limit, precision,
of tablets/capsules, repeated several
of chondroitinases ABC and ACII were
accuracy, recovery, and robustness
times to have a statistical variation,
used for quantitative purposes [11,
tests (see [11]). Furthermore, HPLC
and to calculate the theoretical
14–17]. These methods have been
of the CS disaccharides was used to
CS percentage in each of the food
validated according to the Guidance
evaluate the possible origin of CS in
supplements. The declared CS
for Industry, Bioanalytical Method
nutraceuticals [11, 12, 19] and HPSEC
percentage was found to be from 1.5
Validation from US Department of
was applied for the determination of
up to 47.2 (Table 2).
Health and Human Services, Food
the CS molecular mass parameters
and Drug Administration, Center for
[11–13].
The CS present in the ten nutraceuticals was separated
Table2
Parameters
Food Supplements A
B
C
D
E
F
G
H
I
L
47.2%
16.9%
1.5%
12.4%
24.1%
12.3%
12.3%
30.7%
1.6%
6.0%
Agarose-gel
0.12%
0.34%
1.54%
0.62%
10.20%
4.87%
10.12%
28.50%
1.36%
0.75%
SAX-HPLC
